This invention is concerned with an enzyme technology.
The present invention relates to a novel acylase which deacylates the acyl side chain of a cyclic lipopeptide compound and to a deacylation process comprising the use thereof.
More particularly, this invention relates to a novel acylase which deacylates the acyl side chain of FR901379 Substance, which is produced by Coleophoma sp. F-11899 (FERM BP-2635) (as described in Japanese Kokai Tokkyo Koho H3-184921), or any analog of FR901379 Substance and to a deacylation process using the same.
There has been a standing demand for an acylase capable of deacylating the acyl side chain of a cyclic lipopeptide compound, specifically said FR901379 Substance or an analog thereof, with good efficiency.
The inventors of this invention explored in earnest for a new acylase which might be able to deacylate the acyl side chain of a cyclic lipopeptide compound represented by FR901379 Substance, Echinocandin B and Aculeacin A, the latter two being analogs of FR901379 Substance. As a result, they discovered an acylase in the fermentation broth available upon culture of a certain filamentous fungus and succeeded in achieving the objective deacylation with effectiveness.
The characteristics of the above novel cyclic lipopeptide acylase and of the deacylation process using the enzyme are now described in detail.
The cyclic lipopeptide acylase-producing strain of the invention is first described. The filamentous fungus as a novel cyclic lipopeptide acylase producer specifically includes but is not limited to Oidiodendron sp. No. 30084, Oidiodendron echinulatum IFO 31963, Oidiodendron tenuissimum IFO 6798, Oidiodendron truncatum IFO 9951 and Oidiodendron truncatum IFO 31812, all of which belong to the genus Oidiodendron, and Verticillium sp. No. 30085 which belongs to the genus Verticillium.
The mycological characteristics of Oidiodendron sp. No. 30084 and Verticillium sp. No. 30085 are described below.
The fungus strain No. 30084 was isolated from a soil sample collected in Jouhoku-machi, Higashi Ibaraki-gun, Ibaraki Prefecture. This strain grew repressively on various media, forming colonies varying in color, e.g. greenish gray, brownish orange, yellowish white, etc., according to different kinds of culture media. On several media, strain No. 30084 formed anamorphs showing a conidial structure consisting of a dendritic conidiophore rising up from the surface of the medium and arthroconidia formed at its branches.
The detailed mycological characteristics of strain No. 30084 are as follows.
The cultural characteristics of this fungus on various agar media are summarized in Table 1. Colonies on malt extract agar grew repressively and spread to attain diameters from 1.5 to 2.0 cm after 2 weeks of incubation at 25xc2x0 C. The colony was circular and either raised as a whole or elevated peripherally and depressed in the center. The strain formed anamorphs in abundance, which presented with a powdery surface. The colony was greenish gray with a yellowish gray peripheral zone. The reverse side was light yellow with a yellowish white peripheral zone. On potato dextrose agar, too, the colony grew repressively and spread to attain diameters from 1.5 to 2.0 cm under the same cultural conditions as above. The surface of the colony was centrally elevated or convex, wrinkled, and somewhat felt-like, forming a small amount of anamorphs. The colony was brownish orange to light brown with an orange white peripheral zone. The reverse side was brown with an orange white peripheral zone.
The morphological characteristics of the strain was recorded by observing its growth on Miura""s medium (Miura, H. and M. Kudo: Trans. Mycol. Soc. Japan, 11: 116-118; 1970). The conidiophore of strain No. 30084 stood erect from the surface of the medium and consisted of a tan-colored linear trunk and colorless intricate branches. This dendritic structure made the conidiophore clearly distinguishable from the vegetative hypha. The conidiophore was 90 to 220 (or 240) xcexcm in height, with its trunk being 2 to 3 (or 3.5) xcexcm wide. The branches were bifurcated or trifurcated in succession and spread to occupy the space 30 to 50 xcexcm in height and 40xcx9c60 xcexcm in width over top of the conidiophore. Each branch was fragmented along a plurality of septae, forming conidia each in the form of a rod or an ellipsoid truncated at one end or both ends. The conidium was colorless, smooth-surfaced, unicellular, and varied in size from 2xcx9c4xc3x971.5xcx9c2 xcexcm. Individual conidia were linked through vestiges of cell walls of the branch. The vegetative hypha was smooth-surfaced, septate, colorless, and branched. The hyphal cell was cylindrical and 1.0xcx9c2.5 xcexcm in width. No clamydospore was observed.
Strain No. 30084 was able to grow at 3 to 32xc2x0 C. and the optimum temperature for growth was 24 to 28xc2x0 C. Those data were generated on potato dextrose agar xe2x80x9cNissuixe2x80x9d (Nissui Pharmaceutical).
The foregoing characteristics of strain No. 30084 were compared with the descriptions in the taxonomic reference books on fungi such as (G. R. Barron: The Genera of Hyphomycetes from Soil, pp. 239-241, Williams and Wilkins, Baltimore, 1968), (J. A. von Arx: The Genera of Fungi, Sporulating in Pure Culture, pp. 180-184, J. Cramer, Vaduz, 1974) and (K. H. Domsch, W. Gams and T. H. Anderson: Compendium of Soil Fungi, pp. 517-524, Academic Press, London 1980). As a result, the above characteristics were found to agree with the descriptions of the genus Oidiodendron (Oidiodendron Robak 1932). Therefore, the strain was identified to be a strain belonging to the genus Oidiodendron and named Oidiodendron sp. No. 30084.
The above data are the results of observation after 14 days of incubation at 25xc2x0 C. The color descriptions are based on Methuen Handbook of Colour (Kornerup, A. and J. H. Wanscher, 3rd ed., 525 pp., Methuen, London, 1978).
This strain was originally deposited with National Institute of Bioscience and Human Technology (NIBH, Higashi 1-1-3, Tsukuba-shi, Ibaraki, Japan) (Zip code 305) and assigned with an accession number of FERM P-15550 (date of acceptance: Apr. 2, 1996) but has been converted to a deposit under Budapest Treaty on May 15, 1997 and assigned with an accession number of FERM BP-5943.
The fungus strain No. 30085 was isolated from a soil sample collected in Jouhoku-machi, Higashi Ibaraki-gun, Ibaraki Prefecture. This strain grew repressively on various media, forming colonies varying in color, (e.g. greenish grey, yellowing gray, brown, etc.) and, on several media, produced brown soluble pigments diffusing into the medium. No sporogenic organ was observed normally, but only after prolonged incubation on corn meal agar xe2x80x9cNissuixe2x80x9d (Nissui Pharmaceutical), this strain formed a minimal quantity of asexual spores.
The mycological characteristics of strain No. 30085 are as follows.
The cultural characteristics of the strain on various agar media are summarized in Table 2. The colony on potato dextrose agar grew repressively and spread to attain diameters from 2.0 to 3.0 cm after 2 weeks of incubation at 25xc2x0 C. The colony was circular, flat to elevated, slightly wrinkled and felt-like. An orange-colored exudate was observed on the surface of the colony. The colony was greenish grey to pale gray, with an orangish white to orangish gray peripheral zone. The reverse side was grayish brown to dark brown with a grayish orange peripheral zone and a diffusion of brown soluble pigments into the medium was observed. The colony was Czapek""s solution agar grew much repressively and spread circularly to attain diameters of 1.0xcx9c2.0 cm after 2 weeks of incubation at 25xc2x0 C. The surface of the colony was flat and thin, with no rise-up of aerial hyphae. The surface color was white or yellowish gray, and the reverse color was the same.
The morphologic characteristics of strain No. 30085 were determined according to the findings on said corn meal agar xe2x80x9cNissuixe2x80x9d. The conidiophore of strain No. 30085 was not clearly distinguishable from the vegetative and aerial hyphae, and 2xcx9c5 conidiogenous cells occurred in whirl, or at times singly, at the side of the filament. The conidiogenous cell was colorless, smooths-surfaced, filamentous to elongated flask-shaped (lecythiform), measuring 18 to 37 (45 at times)xc3x971.5 to 2 xcexcm and forming a single conidium to several conidia in a continuous series at the tip. The mode of conidiogenesis appeared to be phialidic but no definite collarette was observed. The conidium was colorless, smooth-surfaced, prolate (ellipsoidal) to bacilliform (rod-shaped), unicellular and 3xcx9c5.5xc3x971.5xcx9c2.5 xcexcm in size. The vegetative hypha was smooth-surfaced, septate, colorless and branched, and although it was usually linear but at times curved and remarkably crimped. The hyphal cell was cylindrical to filiform and 1.0xcx9c5.0 xcexcm in width, containing a large number of intracellular vacuoles. Those vacuoles were released extracellularly with aging of the cell to give a highly viscous exudate. The clamydospore, sclerotium and catenulate form (concatinations) were not observed, but in old culture, the pleurogenesis of a large number of globose cells was at times observed.
Strain No. 30085 was able to grow at 4xcx9c29xc2x0 C., and the optimum temperature for growth was 22xcx9c26xc2x0 C. Those data were generated on potato dextrose agar (Nissui pharmaceutical).
The above characteristics of the strain were compared with the descriptions in several books on the taxonomy of fungi, such as (G. R. Barron: The Genera of Hyphomycetes from Soil, pp. 364, Williams and Wilkins, Baltimore, 1968), (J. A. von Arx: The Genera of Fungi, Sporulating in Pure Culture. pp. 315, J. Cramer, Vaduz, 1974) and (K. H. Domsch, W. Gams and T. H. Anderson: Compendium of Soil Fungi, pp. 859, Academic Press, London, 1980). As a result, strain No. 30085 was found to resemble Verticillium which is a fungus imperfecti (Verticillium Nees 1816). Fungi of the genus Verticillium are different from strain No. 30085 in that the former produce a large number of conidia as a mucoid mass at the tip of the phialide. Regarding this difference, it was considered that strain No. 30085 was able to form only one or a few conidia because of its poor sporogenous ability. Based on the above observation, the strain as identified as a species of the genus Verticillium and named Verticillium sp. No. 30085.
The compositions of malt extract agar, Czapek""s solution agar, and MY20 agar are based on JCM Catalog Nakase, T., 5th ed., 603 p., Japan Collection of Microorganisms and Life Science Research Information Section of the Institute of Physical and Chemical Research, Saitama, 1992).
Those data were generated by observation after 14 days of incubation at 25xc2x0 C. after inoculation. The color descriptions are based on Methuen Handbook of Colour (Kornerup, A. and J. H. Wanscher, 3rd ed., 525 pp., Methuen, London, 1978).
This strain was originally deposited with National Institute of Bioscience and Human Technology (NIBH, Higashi 1-1-3, Tsukuba-shi, Ibaraki, Japan) (Zip code 305) and assigned with an accession number of FERM P-15551 (date of acceptance: Apr. 2, 1996). It was initially designated as Fungus No. 30085. However, the strain was renamed Verticillium sp. No. 30085 on Aug. 16, 1996 and converted to a deposit under Budapest Treaty on May 15, 1997 with assignment of an accession number of FERM BP-5944.
Oidiodendron echinulatum IFO 31963, Oidiodendron tenuissimum IFO 6798, Oidiodendron truncatum IFO 9951 and Oidiodendron truncatum IFO 31812 are subcultures allotted from Institute for Fermentation, Osaka (2-17-85 Juso Hommachi, Yodogawa-ku, Osaka-shi).
The term xe2x80x9ccyclic lipopeptide compoundxe2x80x9d as used throughout this specification means a compound having a polypeptide ring and, as a side chain located on the ring, an xe2x80x9cacylamino groupxe2x80x9d, which substance optionally may have other side chains.
FR901379 Substance, which is a representative species of said xe2x80x9ccyclic lipopeptide compoundxe2x80x9d, is a known compound having antifungal activity as produced by the microorganism Coleophoma sp. F-11899 (FERM BP-2635) (as described in Japanese Kokai Tokkyo Koho H3-184921). It is a compound of the following chemical formula [Ia]: 
The term xe2x80x9canalog of FR901379 Substancexe2x80x9d means any compound of the following general formula [I] or a salt thereof. 
[wherein
R1 is acyl;
R2 is hydroxy or acyloxy;
R3 is hydrogen or hydroxy;
R4 is hydrogen or hydroxy;
R5 is hydrogen or hydroxysulfonyloxy; and
R6 is hydrogen or carbamoyl]
The novel cyclic lipopeptide acylase according to this invention is an acylase derived from a strain belonging to the genus Oidiodendron or the genus Verticillium and capable of deacylating the side chain xe2x80x9cacylaminoxe2x80x9d group of said cyclic lipopeptide compound to an xe2x80x9caminoxe2x80x9d group. To be specific, said acylase is an enzyme which deacylates the palmitoyl side chain of FR901379 Substance or a salt thereof or the acyl side chain of the analog of FR901379 Substance of general formula [I], inclusive of FR901379 Substance, or a salt thereof to give the objective cyclic peptide compound, specifically a compound of the following chemical formula [IIa] (FR179642 Substance): 
or a salt thereof, or an analog of FR179642 of the following general formula [II] which includes FR179642 Substance: 
[wherein R2, R3, R4, R5, and R6 are the same groups as respectively defined above] or a salt thereof.
The preferred species of the above-mentioned salt of compound [I] and [II] are nontoxic mono- or di-salts of the conventional types, thus including metal salts, for example alkali metal salts (e.g. sodium salt, potassium salt, etc.) and alkaline earth metal salts (e.g. calcium salt, magnesium salt, etc.), ammonium salts, salts with organic bases (e.g. trimethylamine salt, triethylamine salt, pyridine salt, picoline salt, dicyclohexylamine salt, N,Nxe2x80x2-dibenzylethylenediamine salt, etc.), organic acid addition salts (e.g. formate, acetate, trifluoroacetate, maleate, tartarate, methanesulfonate, benzenesulfonate, toluenesulfonate, etc.), inorganic acid addition salts (e.g. hydrochloride, hydrobromide, hydroiodide, sulfate, phosphate, etc.) and salts with amino acids (e.g. arginine, aspartic acid, glutamic acid, etc.).
The preferred examples of xe2x80x9clower alkylxe2x80x9d may include straight-chain or branched-chain alkyl groups containing 1xcx9c6 carbon atom(s), such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, isopentyl and hexyl. The more preferred one may be C1-4 alkyl, and the still more preferred one may be methyl.
The preferred example of xe2x80x9chigher alkylxe2x80x9d may include straight-chain or branched-chain alkyl groups containing 7xcx9c20 carbon atom(s), such as heptyl, octyl, 3,5-dimethyloctyl, 3,7-dimethyloctyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl and eicosyl.
The preferred example of xe2x80x9clower alkoxyxe2x80x9d may include straight-chain or branched-chain alkoxy groups such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, pentyloxy, tert-pentyloxy, neopentyloxy, hexyloxy and isohexyloxy.
The preferred example of xe2x80x9chigher alkoxyxe2x80x9d may include straight-chain or branched-chain groups such as heptyloxy, octyloxy, 3,5-dimethyloctyloxy, 3,7-dimethyloctyloxy, nonyloxy, decyloxy, undecyloxy, dodecyloxy, tridecyloxy, tetradecyloxy, hexadecyloxy, heptadecyloxy, octadecyloxy, nonadecyloxy and eicosyloxy.
The preferred examples of xe2x80x9carylxe2x80x9d may include phenyl optionally having lower alkyl (e.g. phenyl, mesityl, tolyl, etc.), naphthyl and anthryl, among others.
The xe2x80x9cacylxe2x80x9d moiety of the preferred species of xe2x80x9cacylaminoxe2x80x9d or xe2x80x9cacylxe2x80x9d may include aliphatic acyl groups derived from carboxylic acids, carbonic acids, carbamic acids, sulfonic acids, etc., aromatic acyl, heterocyclic-acyl, aryl-substituted aliphatic acyl, and heterocyclic-substituted aliphatic acyl.
The preferred examples of said xe2x80x9cacylxe2x80x9d moiety may include aryl (e.g. phenyl, napthyl, anthryl, etc.) which may have one or more (preferably 1xcx9c3) suitable substitutent(s) such as halogen (e.g. fluoro, chloro, bromo, iodo), hydroxy, said higher alkoxy and said aryl; said lower alkoxy; amino; protected amino [preferably acylamino, such as lower alkoxycarbonylamino (e.g. methoxycarbonylamino, ethoxycarbonylamino, propoxycarbonylamino, butoxycarbonylamino, t-butoxycarbonylamino, pentyloxycarbonylamino, hexyloxycarbonylamino, etc.) etc.]; di(lower)alkylamino (e.g. dimethylamino, N-methylethylamino, diethylamino, N-propylbutylamino, dipentylamino, dihexylamino, etc.); lower alkoxyimino (e.g. methoxyimino, ethoxyimino, propoxyimino butoxyimino, t-butoxyimino, pentyloxyimino, hexyloxyimino, etc.); ar(lower)alkoxyimino such as phenyl(lower)alkoxyimino which may have one or more (preferably 1xcx9c3) suitable substituent(s) such as said higher alkoxy (e.g. benzyloxyimino, phenethyloxyimino, benzhydryloxyimino, etc.); heterocyclicthio (preferably pyridylthio) which may have one or more (preferably 1xcx9c3) suitable substituent(s) such as higher alkyl (e.g. heptyl, octyl, 2-ethylhexyl, nonyl, decyl, 3,7-dimethtyloctyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, 3-methyl-10-ethyldodecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, etc.); lower alkanoyl (e.g. formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, hexanoyl, pivaloyl, etc.) which may have one or more (preferably 1xcx9c3) suitable substituent(s) such as heterocyclic group (e.g. thienyl, imidazolyl, pyrazolyl, furyl, tetrazolyl, thiazolyl, thiadiazolyl, etc.) which, in turn, may have one or more (preferably 1xcx9c3) suitable substituent(s) such as amino, said protected amino, said higher alkyl, etc.;
higher alkanoyl (e.g. heptanoyl, octanoyl, nonanoyl, decanoyl, undecanoyl, lauroyl, tridecanoyl, myristoyl, pentadecanoyl, palmitoyl, 10,12-dimethyltetradecanoyl, heptadecanoyl, stearoyl, nonadecanoyl, eicosanoyl, etc.); lower alkenoyl (e.g. acryloyl, methacryloyl, crotonoyl, 3-pentenoyl, 6-hexenoyl, etc.) which may have one or more (preferably 1xcx9c3) suitable substituent(s) such as said aryl which, in turn, may have one or more (preferably 1xcx9c3) suitable substituent(s) such as said higher alkoxy;
higher alkenoyl (e.g. 4-heptenoyl, 3-octenoyl, 3,6-decadienoyl, 3,7,11-trimethyl-2,6,10-dodecatrienoyl, 4,10-heptadecadienoyl, etc.);
lower alkoxycarbonyl (e.g. methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, t-butoxycarbonyl, pentyloxycarbonyl, hexyloxycarbonyl, etc.);
higher alkoxycarbonyl (e.g. heptyloxycarbonyl, octyloxycarbonyl, 2-ethylhexyloxycarbonyl, nonyloxycarbonyl, decyloxycarbonyl, 3,7-dimethylocytyloxycarbonyl, undecyloxycarbonyl, dodecyloxycarbonyl, tridecyloxycarbonyl, tetradecyloxycarbonyl, pentadecyloxycarbonyl, 3-methyl-10-ethyldodecyloxycarbonyl, hexadecyloxycarbonyl, heptadecyloxycarbonyl, octadecyloxycarbonyl, nonadecyloxycarbonyl, eicosyloxycarbonyl, etc.);
aryloxycarbonyl (e.g. phenoxycarbonyl, naphthyloxycarbonyl, etc.);
arylglyoxyloyl (e.g. phenylglyoxyloyl, naphthylglyoxyloyl, etc.);
ar(lower)alkoxycarbonyl which may have one or more suitable substituent(s), for example phenyl(lower)alkoxycarbonyl which may have nitro or lower alkoxy (e.g. benzyloxycarbonyl, phenethyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, etc.);
lower alkylsulfonyl (e.g. methylsulfonyl, ethylsulfonyl, propylsulfonyl, isopropylsulfonyl, pentylsulfonyl, butylsulfonyl, etc.);
arylsulfonyl (e.g. phenylsulfonyl, naphthylsulfonyl, etc.) which may have one or more (preferably 1xcx9c3) suitable substituent(s) such as said lower alkyl, said higher alkoxy, etc.;
ar(lower)alkylsulfonyl such as phenyl(lower)alkylsulfonyl (e.g. benzylsulfonyl, phenethylsulfonyl, benzhydrylsulfonyl, etc.;
said halogen; lower alkyl (e.g. methyl, ethyl, propyl, butyl, t-butyl, pentyl, hexyl, etc.); said higher alkyl; lower alkoxy (e.g. methoxy, ethoxy, propoxy, butoxy, t-butoxy, pentyloxy, hexyloxy, etc.) which may have one or more (preferably 1xcx9c10) suitable substituent(s) such as said lower alkoxy, said halogen, said aryl, etc.; higher alkoxy (e.g. heptyloxy, octyloxy, 2-ethylhexyloxy, nonyloxy, decyloxy, 3,7-dimethyloctyloxy, undecyloxy, dodecyloxy, tridecyloxy, tetradecyloxy, pentadecyloxy, 3-methyl-10-ethyldodecyloxy, hexadecyloxy, heptadecyloxy, octadecyloxy, nonadecyloxy, eicosyloxy, etc.) which may have one or more (preferably 1xcx9c17) suitable substituent(s) such as said halogen; higher alkenyloxy (e.g. 3-heptenyloxy, 7-octenyloxy, 3,6-octadienyloxy, 5-nonenyloxy, 1-decenyloxy, 3,7-dimethyl-6-octenyloxy, 3,7-dimethyl-2,6-octadienyloxy, 8-undecenyloxy, 3,6,8-dodecatrienyloxy, 5-tridecenyloxy, 7-tetradecenyloxy, 1,8-pentadecadienyloxy, 15-hexadecenyloxy, 11-heptadecenyloxy, 7-octadecenyloxy, 10-nonadecenyloxy, 18-eicosenyloxy, etc.); carboxy; said aryl which may have one or more (preferably 1xcx9c3) suitable substituent(s) such as said higher alkoxy; aroyl (e.g. benzoyl, naphthoyl, anthrylcarbonyl, etc.) which may have one or more (preferably 1xcx9c5) suitable substituent(s) such as aryloxy (e.g. phenoxy, naphthyloxy, anthryloxy, etc.) which, in turn, may have one or more (preferably 1xcx9c3) suitable substituent(s) such as, for example, said lower alkoxy or said higher alkoxy; and so on.
Among the above-mentioned species of xe2x80x9cacylxe2x80x9d, the preferred one may be higher alkanoyl, and the particularly preferred one may be palmitoyl.
The xe2x80x9cacylxe2x80x9d moiety in the term of xe2x80x9cacyloxyxe2x80x9d can be referred to aforementioned xe2x80x9cacylxe2x80x9d.
The preferred example of xe2x80x9cacyloxyxe2x80x9d may include lower alkanoyloxy (e.g. formyloxy, acetyloxy, propionyloxy, butyryloxy, isobutyryloxy, valeryloxy, hexanoyloxy, pivaloyloxy, etc.) and phosphonoxy.
The acylase of this invention is one of the so-called inducible enzymes, and it is an essential requisite for the production of the enzyme that a cyclic lipopeptide compound of formula [I], for example FR901379 Substance, is present in the medium in the process of growth of the fungal producer of this acylase. Therefore, this acylase can be produced by culturing a fungal strain capable of producing this particular acylase, for example any of Oidiodendron sp. No. 30084, Oidiodendron echinulatum IFO 31963, Oidiodendron tenuissimum IFO 6798, Oidiodendron truncatum IFO 9951 and Oidiodendron truncatum IFO 31812, all of which belong to the genus Oidiodendron, or Verticillium sp. No. 30085 which belongs to the genus Verticillium, in the presence of said cyclic lipopeptide compound in a culture medium.
More particularly, the acylase can be produced by culturing said acylase-producing fungal strain in a nutrient medium containing one or more assimilable carbon sources and digestable nitrogen sources in the presence of said cyclic lipopeptide compound of formula [I], for example FR901379 Substance, preferably aerobically by, for example, shake culture or submerged culture.
Generally speaking, this novel acylase can be produced by culturing said novel acylase-producing fungus in an aqueous medium containing assimilable carbon and digestable nitrogen sources preferably aerobically by shake culture or submerged culture.
The preferred source of carbon to be present in the culture medium may include carbohydrates such as glucose, xylose, galactose, glycerol, starch and dextrin. As other sources of carbon, there may be mentioned maltose, rhamnose, raffinose, arabinose, mannose, salicin, sodium succinate, etc.
The preferred source of nitrogen may include yeast extract, peptone, gluten meal, cottonseed flour, soybean flour, corn steep liquor, dried yeast, wheat germ, feather powder, peanut flour, etc. and inorganic or organic nitrogen compounds such as ammonium salts, (e.g. ammonium nitrate, ammonium sulfate, ammonium phosphate, etc.) urea, amino acids, etc.
While those sources of carbon and nitrogen are preferably used in suitable combinations, it is not necessary to use pure sources, for even materials of low purity can be used only provided that they contain suitable amounts of growth factors and reasonable amounts of inorganic nutrients. This is because impure materials sometimes contain such growth factors and trace elements and, therefore, can be used with advantage. Optionally, the medium may be supplemented with sodium carbonate or calcium carbonate, sodium phosphate or potassium phosphate, sodium chloride or potassium chloride, sodium iodide or potassium iodide, magnesium salts, copper salts, cobalt salts and other inorganic salts. Particularly, when the culture medium produces a copious foam, an antifoam such as liquid paraffin, a fatty oil, vegetable oil, mineral oil or silicone oil may be added as necessary.
For the high production of this novel acylase, submerged aerobic culture is the preferred methods. For small-scale production, shake culture or surface culture in a flask or bottle is carried out. For culturing the fungus in a large tank, it is preferable to carry out a preculture and inoculate the production tank with the resulting seed culture in order that growth retardation of the fungus may be avoided. Thus, preferably a relatively small quantity of medium is inoculated with spores or hyphae of the microorganism, and the inoculated medium is incubated to prepare a seed culture in the first place, and the resulting seed culture is aseptically transferred to the large tank. The medium for use in this preparation of a seed culture may be substantially identical to or different from the medium for use in the production of the novel acylase.
The agitation and aeration of the fermentation broth can be achieved in various ways. The agitation can be achieved by using a propeller mixer or the like stirring device, rotating or reciprocating a fermentation jar, using a pump of optional construction or plowing sterile air through the medium. The aeration can be achieved by passing sterile air through the fermentation system.
The fermentation is carried out at a temperature of generally about 15xcx9c32xc2x0 C. preferably 20xcx9c30xc2x0 C. and a pH level of 6xcx9c8 for about 50xcx9c150 hours, although those conditions may be varied according to other conditions and scale of fermentation.
The novel acylase thuse produced can be recovered from the fermentation broth by the conventional procedures which are usually employed in the recovery of other known bioactive substances. The novel acylase thus produces occurs in both the cultured mycelium and the supernatant of the broth. Therefore, the novel acylase can be separated from the mycelium and the supernatant or filtrate available on filtration or centrifugation of the fermentation broth and purified by the conventional procedures such as concentration under reduced pressure, freeze-drying, extraction with the common solvent, pH adjustment, treatment with a conventional resin such as an anion exchange resin, a cation exchange resin, a nonionic adsorbent resin, or the like, treatment with a conventional adsorbent such as active charcoal, silicic acid, silica gel, cellulose, alumina, or the like, crystallization, and recrystallization.
Referring to the level of addition of the cyclic lipopeptide compound of the formula [I], e.g. FR9601379 Substance, which is necessary for inducing production of the enzyme, the cyclic lipopeptide compound need be available only in a minimal amount in the medium during the growth of the acylase- producing fungus and usually the objective effect can be well obtained by adding about 0.01 to 1% of the substance to the culture medium. It is not essential to add the cyclic lipopeptide to the preculture medium.
In the fermentative production of FR901379 Substance, it is possible to inoculate the FR901379 production medium with an FR901379-producing strain and said acylase-producing strain concurrently or at staggered times so as to let FR179642 Substance be directly produced in the broth.
The following examples are intended to illustrate but by no means delimit the deacylation technology of the invention which comprises the use of the acylases produced by the fungi Oidiodendron sp. No. 30084, Oidiodendron echinulatum IFO 31963, Oidiodendron tenuissimum IFO 6798, Oidiodendron truncatum IFO 9951, Oidiodendron truncatum IFO 31812 and Verticillium sp. No. 30085.
Process for production of the acylase